1. Field of the Invention
The present invention relates to a method for liquid chromatography of a biological or vital component, and an apparatus therefor.
Particularly, it relates to a method for liquid chromatography of glycosylated hemoglobin, and an apparatus therefor.
2. Related Art Statement
Glycosylated hemoglobins are produced by reaction of a sugar in blood to hemoglobin after the sugar enters erythrocytes, depending on the concentration of the sugar. The concentration of stable-A.sub.lc (s-A.sub.lc) among the glycosylated hemoglobins is said to reflect the average sugar concentration for the past 2 to 3 months. The concentration of s-A.sub.lc is hardly influenced by physiological factors, as compared with blood sugar level, urinary sugar level and the like, and hence is used for the diagnosis of diabetes and as an indication of the progress toward recovery of a diabetic.
A.sub.lc is formed by binding of glucose to the N-terminal of .beta.-chain of hemoglobins. A.sub.lc consists of labile-A.sub.lc (l-A.sub.lc) and stable A.sub.lc (s-A.sub.lc)
Hemoglobin is a conjugated protein having a molecular weight of about 64,000 produced by combination of heme (a pigment) with globin (a protein) and play an important role as an oxygen carrier in the bodies of animals.
When a sugar such as glucose reacts with hemoglobin, release of oxygen becomes difficult, so that supply of oxygen to peripheral blood vessels and tissues becomes difficult. In addition, the structure of the reaction product is fixed, resulting in deformation of blood vessels, etc.
As normal human hemoglobins, those listed in Table 1 are known. The hemoglobin (Hb) of a healthy adult consists of HbA.sub.0, HbA.sub.2 and HbF, and a product formed by reaction with a sugar and HbA.sub.0 is glycosylated hemoglobin (HbA.sub.1). GHb (HbA.sub.1) consists of A.sub.la, A.sub.lb, A.sub.lc and the like which are different in the amino acid structure of hemoglobin and the kind of sugar attached to hemoglobin. Of the glycosylated hemoglobins, A.sub.lc, and by binding of glucose to the N-terminal of .beta. chain, is found in the largest amount.
TABLE 1 __________________________________________________________________________ Hemoglobin Makeup of a Healthy Adult Classification Structure Sugar chain Content __________________________________________________________________________ Hb HbA.sub.0 .alpha..sub.2 .beta..sub.2 -- &gt;90% HbA.sub.2 .alpha..sub.2 .delta..sub.2 -- 2% HbF .alpha..sub.2 .gamma..sub.2 -- &lt;1% GHb HbA.sub.1 A.sub.1 a1 .alpha..sub.2 (.beta.-N-FDP).sub.2 fructose-1,6-diphosphate &lt;1% A.sub.1a2 .alpha..sub.2 (.beta.-N-G6P).sub.2 glucose-6-phosphate &lt;1% A.sub.1b .alpha..sub.2 (.beta.-N-CHO).sub.2 aldehyde &lt;1% A.sub.1c .alpha..sub.2 (.beta.-N-G1c).sub.2 glucose 4.about.6% A.sub.1d ? ? Trace A.sub.1e ? ? Trace __________________________________________________________________________
A.sub.lc is produced non-enzymatically through two reaction steps. In the first reaction, a so-called labile A.sub.lc (l-A.sub.lc) is produced, a part of which reverts to glucose and hemoglobin by a reversible reaction. On the other hand, the second reaction is an irreversible reaction and yields stable-A.sub.lc (s-A.sub.lc).
HbA.sub.l, A.sub.lc, s-A.sub.lc and the like are used as indications of diabetes but it is preferable to measure stable-A.sub.lc (s-A.sub.lc) as a more accurate indication.
As methods for separating glycosylated hemoglobin, hemoglobin, and hemoglobin derivatives, there are many methods in which they are separated on the basis of the difference of their electrical properties. Such methods include electrophoretic methods, ion-exchange chromatographic methods, etc. There are also methods based on high-performance liquid chromatography.
As methods for separating s-A.sub.lc from l-A.sub.lc and measuring the same, there are two methods, i.e., (1) a method in which l-A.sub.lc is previously removed by pretreatment, and (2) a method in which s-A.sub.lc is separated from l-A.sub.lc and detected chromatographically by the use of a separating column.
As the method in which l-A.sub.lc is removed by pretreatment, there are methods in which erythrocytes are incubated with physiological saline or a buffer solution containing semicarbazide and aniline (P. A. Svendsen, et al., Diabetologia, 19, 130 (1980); and D. M. Nathan, et al., Claim. Chem., 28, 512 (1982)). These methods require a troublesome pretreatment or are so poor in rapidity that treatment requires 30 minutes to 4 hours. Commercially available instruments (or apparatus) for exclusive use have an on-line l-A.sub.lc removing mechanism. It has been reported that the treatment time is reduced by adding a commercially available l-A.sub.lc removing reagent to blood and heating the resulting mixture at 50.degree. C. for 1 to 2 minutes (Takahashi et al., Nippon Rinsho Kensa Jidoka Gakkai Kaishi, 12, 133 (1987)). Such a heating treatment is, however, disadvantageous in that it denatures protein to cause precipitation, so that piping or a filter is clogged.
On the other hand, as the method in which s-A.sub.lc is separated from l-A.sub.lc by the use of a separation column, there are, for example, a method using a packing material consisting of silica as base material and a carboxyl group as ion-exchange group, and a method using a packing material consisting of a polyvinyl alcohol type porous resin. Commercially available instruments for exclusive use also permit separation of s-A.sub.lc from l-A.sub.lc.
In the method using a packing material consisting of silica as base material and a carbonyl group as ion-exchange group, a long time of about 20 minutes is required for separating hemoglobin into components A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0 (Jap. Pat. Appln. Kokai (Laid-Open) No. 63-75558).
In the method using a packing material consisting of a polyvinyl alcohol type porous resin, a long time of about 60 minutes is required for separating hemoglobin into components A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0 (Hoshino et al., "Collection of the Substances of Lectures to the 31th Japan Liquid Chromatography Society", No. 29, 175 (1988)).
In another method which uses a vinyl alcohol type copolymer as a packing material, l-A.sub.lc, s-A.sub.lc and HbA.sub.0 are separated from each other in about 25 minutes. This method, however, has not shed light on components A.sub.la, A.sub.lb and HbF (Jap. Pat. Appln. Kokai (Laid-Open) No. 60-213863).
Details of such packing materials have not been clearly reported. It seems that since the particle size of the packing materials and their physical properties such as specific surface area, exchange capacity, etc. have not been made the most suitable, separation of components from each other is not complete or requires a long time.
In the case of a conventional glycosylated hemoglobin analyzer, it takes about 3.5 minutes to separate hemoglobin into five components A.sub.la, A.sub.lb, HbF, A.sub.lc and HbA.sub.0 and it takes 8 minutes to separate hemoglobin into six components A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0 (Takahashi et al., Japan Rinsho Kensa Jidoka Gakkai Kaishi, 12, 133 (1987)). Hemoglobin of all people is not separated into five or six components because A.sub.la, A.sub.lb, HbF or the like is not detected in blood of some people because of low content thereof, and other hemoglobin derivatives are contained in blood of other people. In the present specification, the terms "5-components analysis" and "6-components analysis" are used for convenience.
For separating hemoglobin into the above 5 components, there is used a separation column (.phi.4.6.times.35 mm) using a silica gel type material. On the other hand, for separating hemoglobin into the above 6 components, there is used a separation column having a different size (.phi.4.6.times.120 mm) and using the same material. In both 5-components analysis and 6-components analysis, elution is carried out by a stepwise gradient method in which three eluents are passed through a separating column stepwise. The 5-components analysis and the 6-components analysis are different from each other in a separating column used and the compositions of eluents used. Therefore, the 5-components analysis and the 6-components analysis cannot be carried out in succession. For example, when the 6-components analysis is carried out after the 5-components analysis, the column and eluents should be exchanged and hence troublesome operations are required. Furthermore, after the column exchange, an eluent should be passed through a relief column for about 10 minutes in order to stabilize the column, so that a long time is required for the measurement.
As a packing material packed into such a column, there is usually used a packing material obtained by introducing a carboxymethyl group as functional group into a silica gel with a particle size of about 5 .mu.m as base material.
A separation column prepared by packing a packing material having a particle size of about 5 .mu.m into a .phi.4.6.times.35 mm column permits separation of the above 5 components. However, a separation column having this size (length) does not have a sufficient ability to separate 6 components, and a longer separation column having a inside diameter of 4.6 and a length of 120 mm should be used for this separation.
The chromatographic performances (resolution Rs) are improved in proportion to the square root of the column length (the value of resolution Rs is increased). Thus, the larger the column length, the higher the chromatographic performances but the longer a time required for analysis. In addition, the increase of the column length results in a larger pressure drop and affects the life of column, the life of the seal of a pump, etc.
As described above, in the conventional methods, separation of hemoglobin into 6 components A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0 requires a long time of 8 to 60 minutes. Thus, the conventional methods are disadvantageous with respect to analysis time.
The conventional methods do not make it possible to carry out two analyses for components, i.e., 5-components analysis (separation of hemoglobin into A.sub.la, A.sub.lb, HbF, A.sub.lc and A.sub.0) and 6-components analysis (separation of hemoglobin into A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0) by using a single separation column. That is, they do not make it possible to carry out the 5-components analysis and the 6-components analysis in succession and are disadvantageous with respect to the simplicity of operations and rapidity.
The above prior arts involve a problem in that it takes a long time to separate stable-A.sub.lc (s-A.sub.lc) from other Hb components (A.sub.la, A.sub.lb, HbF, l-A.sub.lc, HbA.sub.0, etc.) and measure the same.
They also involve the following problems. Hemoglobin cannot be separated into 5 components A.sub.la, A.sub.lb, HbF, A.sub.lc and HbA.sub.0, and into 6 components A.sub.la, A.sub.lb, HbF, l-A.sub.lc, s-A.sub.lc and HbA.sub.0, by using a single separation column and eluents having the same individual compositions, and a separation column and eluents should be exchanged, so that troublesome operations are required. The exchange of the separation column and the eluents requires a long time. The prior arts are not economical.